Apparatus and method for determining thyroid function



June 23, 1970 MURTY ET AL 3,516,794

APPARATUS AND METHOD FOR DETERMINING THYROID FUNCTION Filed Dec. 14, 1964 FIG-2 FIG -I INVENTORS. DAS/KA RK MURTY KLAUS FLOREV BY Z 2 A TTORNEV United States Patent 3,516,794 APPARATUS AND METHOD FOR DETERMINING THYROID FUNCTION Dasika R. K. Murty, Kendall Park, and Klaus Florey, Princeton, N.J., assignors, by mesne assignments, to E. R. Squibb & Sons, Inc., New York, N.Y., a corporation of Delaware Filed Dec. 14, 1964, Ser. No. 418,001 Int. Cl. G01n 31/04, 33/16; A61k 27/04 US. Cl. 23230 6 Claims ABSTRACT OF THE DISCLOSURE Method, and apparatus, for determining thyroid function comprising passing a solution of blood serum and radioactive thyroxin or radioactive triiodothyronine onto a mixture of an anion exchanger and inert diluent of cellulose, incubating the solution, squeezing the solution from the anion exchanger, and measuring the percentage radioactivity remaining on the anion exchanger.

This invention relates to a new and improved apparatus and method for determining thyroid function.

Prior to this invention, various kits have been marketed for use in determining thyroid function by means of radioactive substances. These kits generally require the utilization of a standard to be established at the time the test is being performed. One such type of kit entails a particular resin having absorbed thereon either radioactive triiodothyronine or thyroxin. In use, a sample of blood serum (or plasma) is thoroughly mixed with the resin, a portion of the blood sample is then separated from the resin, as by pipetting, and either the residual radioactivity of the resin or the radioactivity of the pipetted blood sample is measured against that of a standard. The ratio of this radioactivity to the radioactivity obtained using a standard, an equal portion of similarly treated blood sample drawn from a person having normal thyroid function determines whether the patient is hyperthyroid, euthyroid (normal) or hypothyroid.

The difliculty with this prior art process, however, resides in the pipetting of the sample of blood. At times, a portion of the finely divided resin was often sucked up into the pipette along with the blood and the radioactivity of the triiodothyronine or thyroxin still adsorbed on such resin, gave an abnormally high radioactive count to the sample.

It is an object of this invention therefore, to provide a simple, improved apparatus for accurately determining thyroid function.

It is another object of this invention to provide a simple, improved method for accurately determining thyroid function without resorting to pipetting techniques. It is a further object of this invention to provide a method of accurately determining thyroid function that will allow the utilization of a table of standards.

The advantages of the present invention will be more readily apparent by reference to the method hereinafter described taken in connection with the accompanying figures in which:

FIG. 1 is a flexible sealed vial; and

FIG. 2 is a kit to be utilized in the practice of this invention.

In accordance with this invention, it has been found that by passing a mixture of a blood sample and a compound seleoted from the group consisting of radioactive triiodothyronine and thyroxin onto a mixture of an anion exchanger and inert diluent, incubating the mixture for a period of time, washing the mixture from the anion exchanger and measuring the percentage resin upice take, eliminates the requirement of pipetting the blood sample and gives a truer, more meaningful determination of thyroid function. This procedure also enables a standard to be set by the supplier rather than by the operator.

In FIG. 1, container 10 has screw cap 11 and plug 12. A mixture of particulate inert diluent and anion exchange resin material 14 are present in the container.

In FIG. 2, container 10 is ready for use and radioactive supply 18 is illustrated as it will be packed for distribution.

The mixture anion exchanger and inert diluent 14 used may be any substance which will adsorb triiodothyronine or thyroxin. Preferably, the resin is an anion exchange resin such as a polymer of styrene quaternary ammonium chloride, which is marketed by Rohm & Haas under the trademark Amber'lite IRA 400. The inert diluent is preferably cellulose. It is utilized in combination with the anion exchange resin to form a homogeneous mixture that will permit a more efficient equilibrium to be achieved between the resin and the serum. The equilibrium referred to herein means the equilibrium established between the radioactive material adsorbed in the serum and that adsorbed by the resin. It has been found that the mixture of resin and inert diluent material having particulate size of from 10 to 200 microns give surprisingly reproducible and accurate results, whereas larger particle sizes give results that are not reproducible, requiring a standard to be made at the same time the actual test is being performed.

Stopper 12 may be cotton or any like material. However, the use of filter paper is to be avoided, as such material will allow particles of resinous material to escape and distort the readings taken by the technician.

Container 10 is preferably made of flexible plastic material (e.g., polyethylene and the like) which is heatsealcd at one end. This seal may be broken by cutting with a scissor or it may have been previously perforated to allow it to be easily torn open. The shape of container 10 may vary in accordance with the knowledge of those skilled in the art. It may be cylindrical or in the form of a funnel, as illustrated in FIG. 1 wherein the top portion is wider than the bottom portion.

The apparatus of this invention is prepared by adding a mixture of particulate resin and particulate cellulose to a container. The ratio of resin to inert diluent being from about 1:30 to about 1:40 with the preferred ratio being from about 1:34 to about 1:36. It is to be understood that this homogeneous inert mixture be sufficient to adsorb therein the quantity of serum utilized.

The particle size of both the anion exchanger and the inert diluent which was found to give the best results were between 10 and 200 microns, with better results being achieved when the average particle sizes were between about 10 to microns. The most favorable results were recorded when the particle size of mixture averaged from between 15 to 30 microns. The inert diluent is of sponge-like character towards blood serum. It readily and uniformly absorbs and expunges the blood sample or serum.

In addition to cellulose, examples of other inert diluents that may be utilized in the practice of this invention are Celite, a siliceous earth (marketed by Johns Manville) and Super Cel.

The radioactive solution of this invention may be selected from the group consisting of radioactive triiodothyronine or thyroxin. The preferred radioactive material is triiodothyronine-I although thyroxin-I thyroxin- I and triiodothyronine-I may also be utilized.

The test is preferably conducted in the presence of a buffering agent, such as one that buffers blood serum in the pH range of about to about 5.5 (and optimally about 5.2 to about 5.3), Although any physiologically accepted buffering agent may be used, the preferred buffers are sodium acetate (0.2 molar, pH 5.5), and trismaleate (0.2 molar, pH 5.2). An aqueous solution of the buffering agent may be added to the blood sample prior to its contact with the anion exchanger present, or the buffer may be first added to the exchanger-holding container, either just prior to use or previously. In the lat ter event, since the apparatus being marketed will be a container, such as a test tube or pliable vial containing particular resin material and solution of the buffering agent, it must be capped to prevent spilling of the buffering solution.

In operation, the sealed container is held by the operator in such a manner that the resin and inert diluent mixture of particles 14 rests above stopper plug 12. Cap 11 is removed and the vessel tapped lightly to allow for all the particles to rest above stopper 12. One cubic centimeter of the serum to be tested is then mixed with one-half cubic centimeter of the radioactive solution 18 provided with the kit. The solution is then poured onto the powdered material mixture in container 10 by cutting the sealed or perforated portion thereof and pouring the solution therein. The bottom of the container 10 is then sealed with cap 11 and incubated for from 45 to 75 minutes at a temperature of from about 20 to degrees centigrade. During this incubation period, a count of the activity in the serium is made in a gamma ray spectrometer such as, Nuclear Chicago Companys Model 1810. Cap 11 after this period of time is removed and about 3 cubic centimeters of water are added and the entire solution is then forced out by any suitable means. For example, if a flexible container is utilized, one may fold or squeeze the container to remove the liquid. Three cubic centimeters of water is again added to container 10, and the above removal process carried out. This washing of the resin is repeated three more times or for a total of about four times. The number of washes may vary depending upon the operators skill and his ability to be consistent. After the washing is completed, the resin material is placed in the counter, and a count again taken. The percentage of resin uptake is then determined by the following formula:

Counts per minute, second count 100 Counts per minute, first count =percentage resin uptake (A) Preparation of flexible container A mixture of 15 milligrams of particulate Amberlite IRA 400 and 5 mg. of particulate cellulose, all particle sizes ranging in sizes from about 10 to about 200 microns, were placed in a flexible container. A small cotton plug was inserted in the mouth of the vial to prevent the particulate material from flowing out when the via was in use.

(B) Tests Twelve serum samples were tested from twelve different patients. The results of these tests are given in Table I. The tests were carired out by adding one cubic centimeters of serum into 0.5 cubic centimeter of a solution of thyroxine-I in an acetate bufler (pH 5.5). The resulting mixture was added to the flexible container and allowed to incubate for about 75 minutes. A radiation count was taken of the contents of the container after about 30 minutes of incubation.

After the incubation period, the mixture and radioactive material were eluted from the container and the particulate material washed three times with 3 milliliters of water each time. A radiation count was made after the final wash.

TABLE I Percent resin Example No. Initial count Residual count uptake The standard for these examples had a percentage resin uptake of from 30 to 35. Therefore, Example 3 illustrates a hyperthyroid condition; Examples 8, 9 and 11 illustrate hypothyroid conditions, whereas the remaining examples would be considered normal or substantially normal.

Similarly, by following the method of Examples 1 to 12, but substituting thyroxin-I triiodothyronine-I and triiodothyronine-I for thyroxin-l similar results are obtainable.

While there have been described various embodiments of the invention, the compositions and methods described are not intended to be understood as limiting the scope of the invention, as it is realized that changes therein are possible and it is further intended that each element recited in any of the following claims is to be understood as referring to all equivalent elements for accomplishing substantially the same results in substantially the same or equivalent manner, it covering the invention broadly in Whatever form its principle may be utilized.

What is claimed is:

1. A test for determining thyroid function which comprises passing a solution of blood serum and a compound selected from the group consisting of radioactive thyroxin and radioactive triiodothyronine onto a mixture of an anion exchanger and an inert diluent of cellulose particulate size about 10 to 200 microns, incubating the solution for a period of time, squeezing the solution from the anion exchanger and measuring the percentage radioactivity remaining on the anion exchanger, according to the formula:

Counts per minute, second count Counts per minute, first count X =percentage resin uptake Counts per minute, second count Counts per minute, first count =percentage resin uptake 3. A test in accordance with claim 2 wherein the radioactive compound is radioactive thyroxin-I 4. A test in accordance with claim 3 wherein the blood sample is blood serum and is admixed with the exchanger in the presence of a buffering agent which buffers the blood serum sample in the pH range of about 5.0 to about 5.5.

5. An apparatus for determining thyroid function Which comprises a flexible elongated container closed at both ends containing a mixture consisting essentially of particulate anion exchange resin and inert diluent of cellulose of particulate size of about 10 to 200 microns in a ratio of from about 1:30 to about 1:40 having adsorbed thereon a compound selected from the group consisting of radioactive thyroxin and radioactive triiodothyronine.

6. An apparatus in accordance with claim 5 wherein the compound is radioactive thyroxin.

References Cited UNITED STATES PATENTS 3,223,619 12/1965 Calmon et al. 21037 6 OTHER REFERENCES Sterling, K., Tabachnick, M.: J. Clin. Endocrin. 21, 456464 (1961).

Siri, W. E.: Isotopic Tracers and Nuclear Radiations, 5 McGraw-Hill, New York, 1949, first edn., pp. 525-527 relied on.

Clayton, J. C., et al.: Biochem. J. 46, 598604 (1950).

MORRIS O. WOLK, Primary Examiner 10 R. M. REESE, Assistant Examiner U.S. Cl. X.R. 

